Thanks carolina, yes blanked by RNA free water used for extraction delivered with the kit. Ethanol and everything needed added a head before the extraction so not a prob.

Not sure how old the kit is but how old is old not to be used?

sure I will not use it but if I do it again what to include or be sure first before extraction.

Can this be caused because I use some how more than 100 mg tissue at the start? 

Hi Temu,

using more than 100 mg of plant material is not the main cause for your problems. Usually, if you use a higher amount than recommended, the column may be clogged or overloaded, resulting in a lower yield.

I never did see a 260/230 ratio below 0 when measuring RNA and 260/280 ratio of 4 also sounds very unusual. Are you sure that the machine is really working fine and you choose the right program for the measurement? Maybe check with a RNA of good quality for which you know the values.

Is the procedure of using the Qiagen plant mini kit established for your samples? Sometimes I work with plant material which is not suitable for this kit. In such cases I first do another method of RNA isolation like CTAB or Trizol, which is enough for applications like cDNA synthesis. If the downstream applications are very sensitive,  use the columns of the Qiagen kit for cleanup of the CTAB- or Trizol-extracted RNA. There is a protocol included in the kits protocol and I always get very high quality RNA with this method.

Good luck!

I have seen this with the nanodrop and the minikit before.  If there is salt remaining, the band shifts over to the left when the sample is in water.  I do three things to clear this up.  1. Always spin dry one extra time.  2. Wash an extra time.  3. Dilute the sample 50:50 in the TE buffer in the kit for measurement and blanking.  There are papers at the nanodrop website that describe this shift.

Hi Temu,

I have been using the same kit and then measured my total RNA using NanoDrop.

Initially, Abs 260/230 reading for some of my samples are ranging from 0.3-1.0. Here are some tips to improve the Abs 260/230 reading:

1) Make sure buffer and centrifugation are done at room temperature

2) During washing, roll the column to make sure that all the surface as well as the column are wash thoroughly.

Other tips which you can also try:

1) Multiple wash to remove residual salt

2) Longer and faster centrifugaton

3) Do not place the sample on ice

4) Make sure that all the buffer has no precipitation prior to use.

After these adjustment/precaution I managed to improve the Abs 260/230 to 2.0

Whilst my Abs 260/280 readings are around 2.0 for both before and after.

GOOD LUCK!